Methods and compositions for administering stem cells

ABSTRACT

The present invention is directed to methods for delivering cells to a target tissue in a mammal using glycoconjugate to traffic the cell to a desired organ in the mammal. The methods according to the present invention are especially applicable to administering stem cells such as those derived from the bone marrow or from umbilical cord tissue. The methods are also useful for targeting a gene of interest to a tissue in a mammal by introducing a cell containing the gene of interest and administering a glycoconjugate to the mammal.

RELATED APPLICATIONS

This application claims priority to, the benefit of, and incorporates byreference for all purposes the following commonly owned patent-relateddocuments, each in its entirety: U.S. provisional patent applicationSer. No. 60/364,498, filed Mar. 15, 2002; and U.S. patent applicationSer. No. 10/388,964, filed 14 Mar. 2003, of which this application is acontinuation.

GOVERNMENT INTEREST

This invention was funded at least in part by funds supplied by the U.S.government. As a result, the U.S. government may have certain rights inthe inventions described herein.

FIELD OF THE INVENTION

The present invention is in the field of clinical medicine and therapy.The invention relates to methods and compositions for targeting cells toan organ of interest.

BACKGROUND OF THE INVENTION

Morell and Ashwell, et al. determined that when a sialyl group ofceruloplasmin is removed by neuraminidase, this plasma protein rapidlydisappears from serum. They disclosed that this phenomenon is due to theuptake by the asialoglycoprotein (ASGP) receptor present in liver cells(J. Biol. Chem., vol. 243:155, (1968)). Thereafter, it was reported thatthe ASGP receptor is present only in liver cells (Adv. Enzymol., vol.41: 99, (1974)). Such specific uptake by liver cells has been identifiedfrom the fact that when asialoceruloplasmin or asialoorosomucoid, whichis experimentally labelled with tritium, is injected into the livingbody, the isotope is selectively detected only in liver cells(Scheinberg I. H., Morell A. G., Stockert R. J.: Hepatic removal ofcirculating proteins. Davidson C. S., ed. Problems in Liver Diseases. pp279-285, New York, Stratton Company, (1979)). In addition, it was alsodisclosed that this receptor specifically recognizes and absorbsglycoproteins having D-galactose or N-acetylgalactosamine as theterminal sugar group (Ann. Rev. Biochem., vol. 51:531, (1982)). The cellmembrane of liver cells comprises a cell structure that combines withasialoglycoprotein terminated with galactose. This cell structure wasfirst named hepato-binding protein (HBP) but is presently calledasialoglycoprotein receptor. Further, it has been observed that amongvarious desialylated glycoproteins, the desialylated alpha(1)-acidglycoprotein, asialoorosomucoid, most rapidly disappears from the serumafter injection. Therefore, it has been determined thatasialo-alpha(1)-acid glycoprotein is both specifically and well taken upby liver cells (J. Biol. Chem., vol. 245:4397 (1970)). Theasialoglycoprotein receptor is constituted with a single polypeptidehaving a molecular weight of about 40,000 and can recognize aglycoprotein having a galactose residue at the nonreductive terminalposition of the saccharide chain (i.e., asialoglycoprotein).

While the physiological functions of an asialoglycoprotein receptor arestill uncertain, it is believed that an asialoglyroprotein receptorparticipates in the metabolism of glycoproteins. In fact, the increaseof the blood level of an asialoglycoprotein is observed in case ofhepatic diseases such as chronic hepatitis, liver cirrhosis and hepaticcancer. Further, the decrease of the quantity of an asialoglycoproteinreceptor is observed in an experimental model of hepatic disorderinduced by administration of chemicals. In view of these phenomena, itmay be possible to diagnose hepatic diseases through assessment of thequantity and quality of an asialoglycoprotein receptor determined by theuse of an asialoglycoprotein-like substance, i.e., an asialoglycoproteinreceptor-directing compound.

Asialoglycoconjugates have been covalently linked to other agents as ameans of targeting chemical (immunosuppressive drugs) and biologicalagents (antibodies) to be taken up by the liver for therapeutic anddiagnostic purposes (see U.S. Pat. Nos. 5,346,696, 5,679,323, and5,089,604)). In addition, localization of bone marrow stem cells andlymphocytes to the liver has been demonstrated (Samlowski, et al., Cell.Immunol., vol. 88:309-322, (1984); Samlowski, et al., Proc. Natl. Acad.Sci., USA, vol. 82:2508-2512, (1985)).

It is also known that a large proportion of cells infused into mammalsadhere to the lung endothelium, independent of cell type orphysiological homing properties. It has been observed that stem cellsaccumulate in the lungs when they are administered (Morrison, et al.,Nature Medicine 2:1281-1282 (1996); Martino, et al., Eur. J. Immunol.,vol. 23:1023-1028 (1993); Pereira, et al., Proc. Natl. Acad. Sci., USA,vol. 92: 4857-4861(1993); and Gao, et al., Cells Tissues Organs, vol.169:12-20 (2001)).

Orosomucoid, asialo-orosomucoid and agalacto/asialo-orosomucoid havebeen shown to inhibit neutrophil activation superoxide anion generation,and platelet activation (Costello, et al., Clin. Exp. Immunol., vol.55:465-472 (1984), and Costello, et al., Nature, vol. 281:677-678(1979)). These proteins also induced transient immunosuppression andprotected against TNF challenge (Bennett, et al., Proc. Natl. Acad. Sci.USA, vol. 77 6109-6113 (1980), and Libert, et al., J. Exp. Med., vol.180:1571-1575 (1994)). Orosomucoid demonstrated specific binding topulmonary endothelial cells, which appeared to be independent ofcarbohydrate recognition sites (Schnitzer, et al., Am. J. Physiol., vol.263:H48-H55 (1992)). Moreover, orosomucoid was shown to bind to skincapillary endothelial cells in a dose dependent manner, therebymaintaining normal capillary permeability in the face of inflammatoryagonists that caused leakage in control animals (Muchitsch, et al.,Arch. Int. Pharmacodyn., vol. 331:313-321 (1996)). Similarly, infusedorosomucoid bound to kidney capillaries and restored the permselectivityof glomerular filtration (Muchitsch, et al., Nephron, vol. 81:194-199(1999)).

A stem cell is a special kind of cell that has a unique capacity torenew itself and to give rise to specialized cell types. Although mostcells of the body such as heart cells or skin cells are committed toconduct a specific function, a stem cell is uncommitted and remainsuncommitted, until it receives a signal to develop into a specializedcell. In 1998, stem cells from early human embryos were first isolatedand grown in culture. It is recognized that these stem cells are,indeed, capable of becoming almost all of the specialized cells of thebody. In recent years, stem cells present in adults also have been shownto have the potential to generate replacement cells for a broad array oftissues and organs, such as the heart, the liver, the pancreas, and thenervous system. Thus, this class of adult human stem cell holds thepromise of being able to repair or replace cells or tissues that aredamaged or destroyed by many devastating diseases and disabilities. Itis highly useful to effect such therapies by targeting stem cells toparticular organs of the body.

In the prior art, adult stem cells generally have been presented to thedesired organs either by injection into the tissue or by infusion intothe local circulation. A need exists to develop methods for delivery ofstem cells through the circulation to specific organs. Such methodswould provide a means to target non-invasively solid organs such as theliver, heart, lungs and kidneys. In addition, very diffuse tissues, suchas the lung, which are not amenable to dosage by injection could betargeted. Such methods would be useful in regenerative stem celltherapies involving such organs as the liver, heart, lungs and kidneys.The present invention addresses these and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention features a method for delivering a cell to atarget tissue in a mammal comprising the steps of administering acarbohydrate-presenting molecule (e.g., a glycoconjugate) to a mammaland then administering the cell to the mammal. As used here, the term“administering” refers to any method of inducing an increasedconcentration of the cell in the circulation of the mammal, whether byinfusion from an extraneous source or by mobilizing the cell into thecirculation from a depot within the mammal, such as the marrow. Meansfor mobilizing stem cells using for example of GM-CSF and GCSF are wellknown in the art (see Simmons, et al., The mobilization of primitivehemopoietic progenitors into the peripheral blood. Stem Cells, vol. 12,Suppl. 1:187-201 (1994)). The methods according to the present inventionare especially applicable to stem cells, such as those derived from thebone marrow, peripheral blood, umbilical cord or from mesenchymal stemcells expanded in culture. The stem cells within the scope of theinvention include any cell capable of differentiating into a desiredtarget tissue. Such cells include pluripotent stem cells, embryonic stemcells, multipotent adult stem cells, and progenitor or precursor cells.

In some embodiments wherein the cell is targeted to the heart, themethods feature administering an orosomucoid (O) or administering anasialoorosomucoid (ASO), and administering the cell to the mammal. Inembodiments wherein the cell is targeted to the lungs, the methodsfeature administering the cell to the mammal in a saline or a serumalbumin-saline solution. In embodiments wherein the cell is targeted tothe liver, the methods feature administering an orosomucoid or anasialoorosomucoid and administering the cell to the mammal. Inembodiments, the orosomucoid is administered concurrently or prior toadministering the cell to the mammal. The methods according to thepresent invention are also useful for either inhibiting or enhancingsequestration of a stem cell in the liver of a mammal even in theabsence of targeting the cell to a target organ.

The glycoconjugates of the present invention may be generallyrepresented by the general formula P-(S)x-Gal wherein P is a peptideresidue of a human serum glycoprotein and S is a sugar residue of ahuman serum glycoprotein, “x” is an integer from 1 to 100, and Gal isgalactose residue. The glycoconjugates may be partially or completelyasialylated. Especially useful glycoconjugates include fetuins,orosomucoids and asialoorosomucoids. The methods of the presentinvention allow cells such as stem cells to be targeted to such targettissues as the heart, the liver, the kidneys and the lungs, amongothers.

The glycoconjugates may be administered to the mammal in any time framerelative to administering the cell. They may be administered before,after or simultaneously with the administration of the cell. In atypical embodiment, the glycoconjugates are administered prior to thecell. The glycoconjugates and the cell may be administered in anysuitable route. In some embodiments, they are administered parenterallyor intravenously to the mammal.

The methods according to the present invention are also useful fortargeting a gene of interest to a tissue in a mammal by introducing acell naturally containing, or a cell transformed with, a gene ofinterest to the mammal. Such methods are useful for treating a diseasecharacterized by a deficiency in a gene product in a mammal byadministering a cell comprising a functional gene encoding the geneproduct into the mammal and administering a glycoconjugate to themammal. According to these methods, a cell containing an exogenousfunctional gene of interest may be administered and localized to aparticular organ in the body where it can function to produce adeficient gene product.

Also, the methods according to the present invention are useful fortreating a disease characterized by tissue damage in a mammal byadministering a cell and administering a glycoconjugate to the mammal.Because stem cells have the potential to generate replacement cells fora broad array of tissues and organs, such as the heart, the pancreas,and the nervous system, stem cells may be targeted to particular organsin the body to repair or replace cells or tissues that are damaged ordestroyed by many devastating diseases and disabilities. In someembodiments, the disease may be a heart disease, a lung disease, akidney disease, or a liver disease, such as, for example, myocardialinfarction, emphysema, cystic fibrosis, microalbuminuria, stroke, orhepatitis.

The methods according to the present invention are also useful fortreating a disease characterized by tissue damage in a mammal byadministering a glycoconjugate to the mammal and administering chemicalsor biopharmaceuticals that mobilize stem cells into the circulation. Theconcentration of circulating mobilized stem cells may be limited becausecertain organs may sequester stem cells, thereby limiting delivery of aneffective dose to the damaged organ. By inhibiting sequestration, theglycoconjugates of the invention increase the cell dose at the organ,thereby increasing the potential to generate replacement cells. Themethods can be used for a broad array of tissues and organs, such as theheart, the pancreas, and the nervous system. Stem cells may be targetedto particular organs in the body to repair or replace cells or tissuesthat are damaged or destroyed by many devastating diseases anddisabilities. In some embodiments, the disease may be a heart disease, alung disease, a kidney disease, a neurological disease, or a liverdisease, such as, for example, myocardial infarction, emphysema, cysticfibrosis, microalbuminuria, stroke, or hepatitis.

In other embodiments, the present invention provides pharmaceuticalcompositions comprising a cell and a glycoconjugate, e.g., glycoprotein.Glycoproteins useful in the present invention include, for example,fetuins, orosomucoids (O), and asialoorosomucoids (ASO). In otheraspects, the present invention features kits for treating tissue damageor for delivering a functional gene or gene product to a tissue in amammal comprising a cell and a glycoprotein. Glycoproteins useful in theinvention include fetuins, orosomucoids, and asialoorosomucoids.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a schematic of liver entrapment of bone marrow stemcells in the liver. Asialoglycodeterminants on the surface of cellsreact with asialoglycoprotein receptors on the surface of hepatocytesresulting in the localization of the bone marrow stem cells in theliver. Glycoconjugates including asialoglycoconjugates, block suchinteractions between asialoglycodeterminants on the surface of cellswith asialoglycoprotein receptors on the surface of hepatocytes.

FIG. 2 shows the carbohydrate structure on two exemplary glycoproteinsof the invention.

FIG. 3 shows the relative binding affinities of different carbohydratesfor the asialoglycoprotein receptor.

FIG. 4 shows the relative binding affinities of different carbohydratesfor the asialoglycoprotein receptor.

DETAILED DESCRIPTION OF THE INVENTION

A. Introduction

The present invention is directed to methods for delivering a cell to atarget tissue in a mammal. The methods comprise the steps ofadministering, either simultaneously or sequentially, acarbohydrate-presenting molecule (i.e., a “glycoconjugate”) and a cellto the mammal. In the methods of the present invention, glycoconjugates,especially asialoglycoconjugates, including asialo plasma proteins suchas asialoorosomucoid (asialo alpha-(1) -acid glycoprotein), are thoughtto transiently bind the hepatic asialoglycoprotein receptor and therebycompetitively inhibit attachment of cells bearing asialodeterminantsfrom these receptors. Without wishing to be bound by theory,hyposialylated and desialyated proteins/glycoconjugates (also calledasialoglycoconjugates) and cells which bear similar determinants arebound or “trapped” in the liver as a consequence of binding to thehepatic asialoglycoprotein receptors (see FIG. 1). Occupation of thereceptor by the asialoglycoconjugate inhibits sequestration of the cellsbearing similar determinants of interest in the liver.

In addition, the present disclosure shows that glycoconjugates of theinvention prevent infused cells from concentrating in the alveolarvasculature. This finding suggests that lung sequestration of the cellsmay be related to expression of inflammatory receptors on endothelialcells, analogous to the reperfusion syndrome (see, e.g., Kilgore, etal., Cardiovasc. Res., vol. 28:437-444 (1994), and Eror, et al., Clin.Immunol., vol. 90:266-275 (1999)). This is supported by reports thatorosomucoid, ASO, and agalacto/asialo-orosomucoid inhibit neutrophilactivation superoxide anion generation, as well as platelet activation,as noted above.

The present invention further demonstrates that the glycoproteins may beused to traffic or target cells to particular organs of the body byaltering the particular glycoconjugate administered. The present methodsare useful to improve the efficacy of bone marrow and stem celltransplants, tissue repair, or gene therapy.

In embodiments wherein the cell is targeted to the lungs, the methodsfeature administering the cell to the mammal in a saline or serumalbumin-saline solution. In some embodiments wherein the hematopoieticstem cell is targeted to the heart, the methods feature administering anasialoorosomucoid, and administering the cell to the mammal. In otherembodiments wherein the mesenchymal stem cell is targeted to the heart,the methods feature administering an orosomucoid, and administering thecell to the mammal. In embodiments wherein the hematopoietic stem cellis targeted to the liver, the methods feature administering anorosomucoid and administering the cell to the mammal. In otherembodiments wherein the mesenchymal stem cell is targeted to the liver,the methods feature administering an asialoorosomucoid and administeringthe cell to the to the mammal. In some embodiments, the orosomucoid orasialoorosomucoid is administered in at least two infusions prior toadministering the cell to the mammal. The methods according to thepresent invention are also useful for inhibiting sequestration of a cellin the liver of a mammal even in the absence of targeting the cell to atarget organ.

Asialoglycoconjugates, for example, asialofetuin and other asialo plasmaproteins, are able to bind to the hepatic parenchymal and Kupffer cellasialoglycoprotein receptors. Blocking these receptors from binding andtrapping cells bearing asialodeterminants, such as bone marrow cells,facilitates and increases the interval of their systemic circulation. Inthe case of bone marrow stem cells the administration of these compoundsprevents the loss and destruction of bone marrow stem cells andincreases the efficiency of engraftment. Bone marrow cells have cellsurface asialodeterminants capable of binding to the asialoglycoproteinreceptor, and this binding can be inhibited by the application ofasialoglycoproteins.

The present invention takes advantage of the observation that when humanperipheral hematopoietic stem (CD34+) cells or mesenchymal stem cellsare infused into the jugular vein of immunodeficient mice, they localizepredominantly in the lungs. When the cells are preceded by an infusionof asialoorosomucoid, the hematopoietic stem cells predominantlylocalize in the heart, whereas the mesenchymal stem cells localize inthe liver. Alternately, when the cells are preceded by an infusion oforosomucoid (0), the hematopoietic stem cells localize in the liver,whereas the mesenchymal stem cells predominantly localize in the heart.

These protein infusions cause a more quantitative localization into thespecific organs than occurs without them. Furthermore, hematopoieticstem cells that localize in the heart due to the influence ofasialoorosomucoid leave the vascular space and are observed among thecardiac muscle cells by one hour after infusion. Moreover, once in thetissue, these cells lose their CD34 antigen, indicating that they are inthe process of differentiating into cardiomyocytes. Additionally, at onehour CD34+ cells have been demonstrated to move from the vasculatureinto lung tissue. In an orosomucoid-treated mouse, clusters of stemcells are found in the liver parenchyma and are also demonstrated tolose their CD34 antigen, again suggesting differentiation intohepatocytes.

The present invention demonstrates the ability to direct highconcentrations of stem cells to a specific organ in an atraumaticmanner. This enhances the probability and the rate at which stem cellsmigrate into a target tissue and differentiate into the desired celltype. The present invention utilizes the observation that delivery oforosomucoid or ASO to the vessel proximal to the heart causes transfusedstem cells to accumulate in the heart. Without wishing to be bound bytheory, the effect may be caused by the glycoprotein infusionsensitizing the endothelium directly downstream from the infusion site,which causes the endothelial cells to bind stem cells and enhance theirmigration across the endothelium into the tissue.

The present findings with glycoconjugates indicate that the majority ofa stem cell transfusion can be concentrated in the target organ, therebyproviding the means to deliver an effective regimen of cell doses. Thisoffers an opportunity to non-invasively target solid organs such as theheart, thereby competing with invasive direct injection. Perhaps moreimportantly, glycoconjugates provide the means to target very diffusetissues, such as the liver and the kidney, which are not amenable todosage by injection.

It is recognized that hematopoietic stem cells (HSC) recovered from themarrow, peripheral blood or umbilical cord blood and mesenchymal stemcells (MSC) recovered as marrow stromal cells, stromal cells fromliposuction fat, or proliferated from stationary stromal progenitorcells in cord blood-depleted expelled placentas appear to be almostinterchangeable in their differentiation ability, and act as multipotentstem cells.

Such cells have been shown to differentiate into functional cells whenlocalized in specific organs and tissues: hepatocytes and cholangiocytesin the liver; cardiac muscle cells and arterial smooth muscle cells andendothelial cells in the heart; pneumocytes I and II in alveoli andbronchial epithelium in the lungs; chondrocytes for cartilagerestoration; and intestinal mucosal cells, etc.

B. Stem Cells

Stem cells may hold the key to replacing cells lost in many devastatingdiseases such as Parkinson's disease, diabetes, acute and chronic heartdisease, end-stage kidney disease, liver failure, and cancer. For manydiseases, there are no effective treatments but the goal is to find away to replace what natural processes have taken away.

To date, published scientific papers indicate that adult stem cells havebeen identified in brain, bone marrow, peripheral blood, blood vessels,skeletal muscle, epithelia of the skin and digestive system, cornea,dental pulp of the tooth, retina, liver, and pancreas. Thus, adult stemcells have been found in tissues that develop from all three embryonicgerm layers.

By way of definition, the following terms are understood in the art:

A “stem cell” is a cell from the embryo, fetus, or adult that has, undercertain conditions, the ability to reproduce itself for long periods or,in the case of adult stem cells, throughout the life of the organism. Italso can give rise to specialized cells that make up the tissues andorgans of the body.

A “pluripotent stem cell” has the ability to give rise to types of cellsthat develop from the three germ layers (mesoderm, endoderm, andectoderm) from which all the cells of the body arise. The only knownsources of human pluripotent stem cells are those isolated and culturedfrom early human embryos and from fetal tissue that was destined to bepart of the gonads.

An “embryonic stem cell” is derived from a group of cells called theinner cell mass, which is part of the early (4- to 5-day) embryo calledthe blastocyst. Once removed from the blastocyst the cells of the innercell mass can be cultured into embryonic stem cells. These embryonicstem cells are not themselves embryos.

An “adult stem cell” is an undifferentiated (unspecialized) cell thatoccurs in a differentiated (specialized) tissue, renews itself, andbecomes specialized to yield all of the specialized cell types of thetissue in which it is placed when transferred to the appropriate tissue.Adult stem cells are capable of making identical copies of themselvesfor the lifetime of the organism. This property is referred to as“self-renewal.” Adult stem cells usually divide to generate progenitoror precursor cells, which then differentiate or develop into “mature”cell types that have characteristic shapes and specialized functions,e.g., muscle cell contraction or nerve cell signalling. Sources of adultstem cells include bone marrow, blood, the cornea and the retina of theeye, brain, skeletal muscle, dental pulp, liver, skin, the lining of thegastrointestinal tract, and pancreas.

Stem cells from the bone marrow are the most studied type of adult stemcells. Currently, they are used clinically to restore various blood andimmune components to the bone marrow via transplantation. There arecurrently identified two major types of stem cells found in bone marrow:hematopoietic stem cells (HSC, or CD34+ cells) which are typicallyconsidered to form blood and immune cells, and stromal (mesenchymal)stem cells (MSC) that are typically considered to form bone, cartilage,muscle, and fat. However, both types of marrow-derived stem cellsrecently have demonstrated extensive plasticity and multipotency intheir ability to form the same tissues.

The marrow, located in the medullary cavity of bones, is the sole siteof hematopoiesis in adult humans. It produces about six billion cellsper kilogram of body weight per day. Hematopoietically active (red)marrow regresses after birth until late adolescence after which time itis focused in the lower skull vertebrae, shoulder and pelvic girdles,ribs, and sternum. Fat cells replace hematopoietic cells in the bones ofthe hands, feet, legs and arms (yellow marrow). Fat comes to occupyabout fifty percent of the space of red marrow in the adult and furtherfatty metamorphosis continues slowly with aging. In very oldindividuals, a gelatinous transformation of fat to a mucoid material mayoccur (white marrow). Yellow marrow can revert to hematopoieticallyactive marrow if prolonged demand is present such as with hemolyticanemia. Thus, hematopoiesis can be expanded by increasing the volume ofred marrow and decreasing the development (transit) time from progenitorto mature cell.

The marrow stroma consists principally of a network of sinuses thatoriginate at the endosteum from cortical capillaries and terminate incollecting vessels that enter the systemic venous circulation. Thetrilaminar sinus wall is composed of endothelial cells, anunderdeveloped, thin basement membrane, and adventitial reticular cellsthat are fibroblasts capable of transforming into adipocytes. Theendothelium and reticular cells are sources of hematopoietic cytokines.Hematopoiesis takes place in the intersinus spaces and is controlled bya complex array of stimulatory and inhibitory cytokines, cell-to-cellcontacts, and the effects of extracellular matrix components onproximate cells. In this unique environment, lymphohematopoietic stemcells differentiate into all of the blood cell types. Mature cells areproduced and released to maintain steady-state blood cell levels. Thesystem may meet increased demands for additional cells as a result ofblood loss, hemolysis, inflammation, immune cytopenias, and othercauses. The engraftment efficiency of bone marrow stem cells could beimproved by preventing entrapment by the liver via the hepaticasialoglycoprotein receptor.

A “progenitor” or “precursor” cell occurs in fetal or adult tissues andis partially specialized; it divides and gives rise to differentiatedcells. Researchers often distinguish precursor/progenitor cells fromadult stem cells in that when a stem cell divides, one of the two newcells is often a stem cell capable of replicating itself again. Incontrast, when a progenitor/precursor cell divides, it can form moreprogenitor/precursor cells or it can form two specialized cells.Progenitor/precursor cells can replace cells that are damaged or dead,thus maintaining the integrity and functions of a tissue such as liveror brain.

Means for isolating and culturing stem cells useful in the presentinvention are well known. Umbilical cord blood is an abundant source ofhematopoietic stem cells. The stem cells obtained from umbilical cordblood and those obtained from bone marrow or peripheral blood appear tobe very similar for transplantation use. Placenta is an excellentreadily available source for mesenchymal stem cells. Moreover,mesenchymal stem cells have been shown to be derivable from adiposetissue and bone marrow stromal cells and speculated to be present inother tissues. While there are dramatic qualitative and quantitativedifferences in the organs from which adult stem cells can be derived,the initial differences between the cells may be relatively superficialand balanced by the similar range of plasticity they exhibit. Forinstance, adult stem cells both hematopoietic and mesenchymal, under theappropriate conditions can become cardiac muscle cells. Delineation offull range of potential for adult stem cells has just begun.

Stem cells may be isolated for transduction and differentiation usingknown methods. For example, in mice, bone marrow cells are isolated bysacrificing the mouse and cutting the leg bones with a pair of scissors.Stem cells may also be isolated from bone marrow cells by panning thebone marrow cells with antibodies which bind unwanted cells, such asCD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1 (granulocytes), andlad (differentiated antigen presenting cells). For an example of thisprotocol, see Inaba, et al., J. Exp. Med., vol. 176:1693-1702(1992).

In humans, CD34+ hematopoietic stem cells can be obtained from a varietyof sources, including cord blood, bone marrow, and mobilized peripheralblood. Purification of CD34+ cells can be accomplished by antibodyaffinity procedures. An affinity column isolation procedure forisolating CD34+ cells is described by Ho, et al., Stem Cells, vol. 13(suppl. 3): 100-105(1995). See also, Brenner, Journal of Hematotherapy,vol. 2: 7-17 (1993). Methods for isolating, purifying, and culturallyexpanding mesenchymal stem cells are known. Specific antigens for MSCare also known (see, e.g., U.S. Pat. Nos. 5,486,359 and 5,837,539).

C. Carbohydrate Presenting Molecules

The carbohydrate-presenting molecules useful in the present inventioncan be any molecule capable of presenting the appropriate carbohydratestructure that leads to enhancing or inhibiting the targeting of thecell of interest to a target tissue. The targeting function can becarried out using a carbohydrate molecule such as an oligosaccharide,polysaccharide, or the carbohydrate structure can be bound to largermolecule or carrier, referred to here as a glycoconjugate. Typically,the carbohydrate molecule will be linked to either a naturally occurringcarrier (e.g., as part of a glycoprotein or glycolipid) or the carriermay be synthetic (e.g., an engineered polypeptide sequence). One ofskill will recognize that a number of carriers can be used to presentthe appropriate structure. Examples of appropriate carrier moleculesinclude polypeptides, lipids, and the like. Preparation and use oftargeted compounds using asialo carbohydrate moieties is described inthe art (see, e.g., U.S. Pat. Nos. 5,679,323, 5,089,604, 5,032,678, and5,284,646). One of skill will recognize that such compounds can also beused as carbohydrate presenting molecules useful in the presentinvention.

In cases in which the glycoconjugate is a glycoprotein it may begenerally represented by the general formula P-(S)x-Gal, wherein “P” isa peptide residue of a human serum glycoprotein and “S” is a sugarresidue of a human serum glycoprotein, “x” is an integer from 1 to 100,and “Gal” is a galactose residue. Especially useful glycoconjugatesinclude fetuins and asialofetuins (see FIG. 2), orosomucoids andasialoorosomucoids, and galactose-bonded polylysine, galactose-bondedpolyglucosamine, and the like.

The methods of the present invention allow cells such as stem cells tobe targeted to such target tissues as the heart, the liver, the kidneys,and the lungs, among others. Parenteral administration of aglycoconjugate, such as asialoorosomucoid, may be used to block thehepatic asialoglycoprotein receptor and allow the cells bearing surfaceasialodeterminants (for example, peanut agglutinin (PNA)+ cells) tocontinue to circulate and migrate to the marrow space. Asialoorosomucoidis one of the glycoproteins that has been shown to bind to the hepaticasialoglycoprotein receptor and has been extensively used tocharacterize this receptor.

Different compounds have different binding affinities for theasialoglycoprotein receptor, depending upon the carbohydrate presented(see, FIGS. 3 and 4). Thus, one of skill can modulate cell targeting byusing compounds that present different carbohydrate structures.

Intravenous administration of a glycoconjugate, especially anasialoglycoprotein such as asialoorosomucoid, may be used to block thehepatic asialoglycoprotein receptor and allow the cells bearing surfaceasialodeterminants to continue to circulate and migrate to the marrowspace or to the organ of interest. The glycoconjugates may beadministered to the mammal in any time frame relative to the cells, butin some embodiments, the glycoconjugates are administered prior toadministering the cell. The asialoglycoconjugates and the cell may beadministered in any suitable route, but in some embodiments, they areadministered intravenously to the mammal, and in other embodiments, theyare administered parenterally. In embodiments wherein the cell istargeted to the lungs, the methods feature administering the cell to themammal in a saline or serum albumin-saline solution. In some embodimentswherein the hematopoietic stem cell is targeted to the heart, themethods feature administering an asialoorosomucoid and administering thecell to the mammal. In other embodiments wherein the mesenchymal stemcell is targeted to the heart, the methods feature administering anorosomucoid and administering the cell to the mammal. In embodimentswherein the hematopoietic stem cell is targeted to the liver, themethods feature administering an orosomucoid and administering the cellto the mammal. In other embodiments wherein the mesenchymal stem cell istargeted to the liver, the methods feature administering anasialoorosomucoid and administering the cell to the to the mammal. Insome embodiments, the orosomucoid or asialoorosomucoid is administeredin at least two infusions, prior to and after administering the cell tothe mammal. The methods according to the present invention are alsouseful for inhibiting sequestration of a cell in the liver of a mammaleven in the absence of targeting the cell to a target organ.

The alpha-(1)-acid glycoprotein (orosomucoid or AAG) is a normalconstituent of human plasma (650±215 μg/ml) that increases inconcentration as much as five-fold in association with acuteinflammation and cancer, and thus is recognized as an acute phaseprotein. Orosomucoid consists of a single polypeptide chain, has amolecular weight of 44,100, and contains approximately 45% carbohydrateincluding 12% sialic acid. It is the most negatively charged of theplasma proteins. Certain of the biological properties of orosomucoid arerelated to its sialic acid content. Thus, clearance and immunogenicityof orosomucoid are markedly increased on desialylation. The biologicalfunctions of orosomucoid are largely unknown. Orosomucoid has theability to inhibit certain lymphocyte reactivities includingblastogenesis in response to concanavalin A, phytohaemagglutinin andallogeneic cells, and these inhibitory effects are enhanced inassociation with desialylation. It has been reported thatunphysiologically large (5-15 mg/ml) amounts of orosomucoid inhibit theplatelet aggregation induced by ADP and adrenaline, and there isevidence that a sialic acid-deficient species of orosomucoid appearselevated in several chronic disease states.

D. Gene Therapy

The present invention is also directed to using living cells to delivertherapeutic genes into the body. In some embodiments, the therapeuticgene is a transgene. For example, the delivery cells—a type of stemcell, a lymphocyte, or a fibroblast—are removed from the body, and atherapeutic transgene is introduced into them via vehicles well known tothose skilled in the art such as those used in direct gene transfermethods. While still in the laboratory, the genetically modified cellsare tested and then allowed to grow and multiply and, finally, areinfused back into the patient. Alternatively, allogeneic cells that bearnormal, endogenous genes can reverse a deficiency in a particular targettissue. Use of cells bearing either transgenes or normal, endogenousgenes is referred to herein as gene therapy.

Gene therapy using genetically modified cells offers several uniqueadvantages over direct gene transfer into the body. First, the additionof the therapeutic transgene to the delivery cells takes place outsidethe patient, which allows the clinician an important measure of controlbecause they can select and work only with those cells that both containthe transgene and produce the therapeutic agent in sufficient quantity.

Of the stem cell-based gene therapy trials that have had a therapeuticgoal, approximately one-third have focused on cancers (e.g., ovarian,brain, breast myeloma, leukemia, and lymphoma), one-third on humanimmunodeficiency virus disease (HIV-1), and one-third on so-calledsingle-gene diseases (e.g., Gaucher's disease, severe combined immunedeficiency (SCID), Fanconi anemia, Fabry disease, and leukocyteadherence deficiency).

In view of the foregoing, the methods according to the present inventionare useful for targeting a gene of interest (either a transgene or anendogenous gene) to a tissue in a mammal by introducing a cellcomprising the gene of interest and administering a glycoconjugate tothe mammal. Such methods are useful for treating a disease characterizedby a deficiency in a gene product in a mammal by administering a cellcomprising a functional gene encoding the gene product into the mammaland administering a glycoconjugate to the mammal. Stem cells may be usedas a vehicle for delivering genes to specific tissues in the body. Stemcell-based therapies are a major area of investigation in cancerresearch.

The current invention provides localizing of transfused cells such asstem cells to provide a functional gene to a patient suffering from adisease caused by a lack of that gene. In many instances of geneticallybased diseases, a low level production of that gene product willeffectively ameliorate or cure the disease. By providing the gene thatis deficient through transfusion of stem cells from a normal donor intothe patient, the stem cells may be directed to localize in an organ ortissue of choice, causing a microchimerization of that patient in thatorgan or tissue, from which organ or tissue that gene product can bedelivered to the patient. Therefore, the present invention provides theability to direct the localization of the transfused cells such asallogeneic stem cells that have a stable, normal gene. Such transfusedcells then create a stable micro-chimera of the recipient.

Those of skill in the art are aware of the genetic deficienciescausative of a large array of genetically based diseases. Exemplarygenes and diseases that can be treated include CTFR protein in cysticfibrosis and proteins associated with coagulopathy in the liver. Forexample, treatment of Hemophilia A can be accomplished using genetherapy. In such embodiment, a transfusion of such cells as umbilicalcord blood hematopoietic stem cells may be administered to deliver anintact normal Factor VIII gene. Alternatively, transformed cells cancomprise a normal, wild-type Factor VIII gene. Such cells carrying afunctional Factor VII gene may be directed to localize in the liver,preferably by orosomucoid or asialoorosomucoid perfusion prior to theinfusion of the stem cells. The cells transform into hepatocytes andbegin secreting Factor VIII into the blood.

Other embodiments of gene therapy according to the present inventioninclude treating Hemophilia B (Factor IX deficiency), and antithrombinIII, Protein C, and Protein S deficiencies. While these diseases allinvolve the blood coagulation system, gene therapy may include treatingdifferent tissues, such as muscular dystrophy, cystic fibrosis, and thelike.

E. Introducing Transgenes Into Stem Cells

Means for introducing transgenes into cells are well known. A variety ofmethods for delivering and expressing a nucleic acid within a mammaliancell are known to those of ordinary skill in the art. Such methodsinclude, for example, viral vectors, liposome-based gene delivery (WO93/24640; Mannino Gould-Fogerite, BioTechniques, vol. 6(7):682-691(1988); U.S. Pat. No. 5,279,833; WO 91/06309; Felgner, et al., Proc.Natl. Acad. Sci. USA, vol. 84:7413-7414 (1987); and Budker, et al.,Nature Biotechnology, vol. 14(6):760-764 (1996)). Other methods known tothe skilled artisan include electroporation (U.S. Pat. Nos. 5,545,130,4,970,154, 5,098,843, and 5,128,257), direct gene transfer, cell fusion,precipitation methods, particle bombardment, and receptor-mediateduptake (U.S. Pat. Nos. 5,547,932, 5,525,503, 5,547,932, and 5,460,831).See also U.S. Pat. No. 5,399,346.

Widely used retroviral vectors include those based upon murine leukemiavirus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immunodeficiencyvirus (SIV), human immunodeficiency virus (HIV), and combinationsthereof. See, e.g., Buchscher, et al., J. Virol., vol. 66(5):2731-2739(1992); Johann, et al., J. Virol., vol. 66(5):1635-1640 (1992);Sommerfelt, et al., Virol., vol. 176:58-59 (1990); Wilson, et al., J.Virol., vol. 63:2374-2378 (1989); Miller, et al., J. Virol., vol.65:2220-2224 (1991); PCT/US94/05700, and Rosenburg and Fauci, inFundamental Immunology, Third Edition (Paul ed., 1993)).

AAV-based vectors are also used to transduce cells with target nucleicacids, e.g., in the in vitro production of nucleic acids andpolypeptides, and in vivo and ex vivo gene therapy procedures. See West,et al., Virology, vol. 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO93/24641; Kotin, Human Gene Therapy, vol. 5:793-801 (1994); Muzyczka, J.Clin. Invest., vol. 94:1351 (1994), and Samulski (supra) for an overviewof AAV vectors. Construction of recombinant AAV vectors are described ina number of publications, including Lebkowski, U.S. Pat. No. 5,173,414;Tratschin, et al., Mol. Cell. Biol., vol. 5(11):3251-3260 (1985);Tratschin, et al., Mol. Cell. Biol. Vol. 4:2072-2081 (1984); Hermonatand Muzyczka, Proc. Natl. Acad. Sci., USA, vol. 81:6466-6470 (1984); andSamulski, et al., J. Virol., vol. 63:03822-3828 (1989).

Retroviral vectors are typically used for cells useful in the presentinvention. Such vectors may comprise, for example, an HIV-2 packageablenucleic acid packaged in an HIV-2 particle, typically using a packagingcell line. Cell transduction vectors have considerable commercialutility as a method of introducing genes into target cells. Inparticular, gene therapy procedures, in which the cell transductionvectors of the invention are used to transduce target cells with atherapeutic nucleic acid in an in vivo or ex vivo procedure, may beused. Gene therapy provides a method for combating chronic diseasescaused by a gene deficiency, infectious diseases such as HIV, as well asnon-infectious diseases such as cancer.

Stem cells such as CD34+ stem cells may be used in ex vivo proceduresfor cell transduction and gene therapy. The present invention utilizesthe feature that stem cells differentiate into other cell types invitro, or can be introduced into a mammal (such as the donor of thecells) where they will engraft in the bone marrow unless targeted toanother organ for differentiation. Hence, the present invention extendsto directing stem cells to particular organs to regenerate tissue suchas to the heart to regenerate cardiac muscle cells, to the lung toregenerate alveoli, and to the kidneys to regenerate tissue and todirecting cells such as CD34+ stem cells to an organ to ameliorate agenetic abnormality by providing efficacious amounts of a deficient geneproduct. Methods for differentiating CD34+ cells in vitro intoclinically important immune cell types using cytokines such a GM-CSF,IFN-γ and TNF-α are known (see Inaba, et al., J. Exp. Med., vol. 176,1693-1702(1992), and Szabolcs, et al., 154: 5851-5861(1995)). Yu, et al.(Proc. Natl. Acad. Sci., USA, vol. 92: 699-703(1995)) describe a methodof transducing CD34+ cells from human fetal cord blood using retroviralvectors.

F. Pharmaceutical Compositions

In other embodiments, the present invention provides pharmaceuticalcompositions comprising a cell and a glycoconjugate of the invention.Exemplary glycoproteins include orosomucoids and asialoorosomucoids. Inother aspects, the present invention features kits for treating tissuedamage or for delivering a functional gene or gene product to a tissuein a mammal comprising a cell and a glycoprotein. Stem cells generallyhave been presented to the desired organs either by injection into thetissue, by infusion into the local circulation, or by mobilization ofautologous stem cells from the marrow accompanied by prior removal ofstem cell-entrapping organs before mobilization when feasible, e.g.,splenectomy.

Glycoconjugates may be administered prior to, concomitantly with, orafter infusing the stem cells. In some embodiments, an intravenous fluidbag may be used to administer the glycoconjugate in a saline or dextrosesolution with and without protein, or serum-free media, including, butnot restricted to, RPMI 1640 or AIM-V. In such embodiments, theglycoconjugate may be mixed with the cells in the same bag or in a“piggy-back”. The glycoconjugate may also be continued afteradministration of the cells to permit longer systemic circulation timesor increased specific organ accumulation. This procedure may be repeatedas often as needed for delivering a therapeutic dose of the cells to thetarget organ. The preparation may be used with little concern fortoxicity given data from animal studies demonstrating no side effects atdoses of 3-7 mg of glycoconjugate per ml of blood volume.

Administration of cells transduced ex vivo can be by any of the routesnormally used for introducing a cell or molecule into ultimate contactwith blood or tissue cells. The transduced cells may be administered inany suitable manner, preferably with pharmaceutically acceptablecarriers. Suitable methods of administering such cells in the context ofthe present invention to a patient are available, and, although morethan one route can be used to administer a particular composition, aparticular route can often provide a more immediate and more effectivereaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there are a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.Parenteral administration is one useful method of administration. Theformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and in some embodiments, can bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example, water, forinjections, immediately prior to use. These formulations may beadministered with factors that mobilize the desired class of adult stemcells into the circulation.

Extemporaneous injection solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.Cells transduced by the vector as described above in the context of exvivo therapy can also be administered parenterally as described above,except that lyophilization is not generally appropriate, since cells aredestroyed by lyophilization.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular cells employed and the condition of thepatient, as well as the body weight of the patient to be treated. Thesize of the dose also will be determined by the existence, nature, andextent of any adverse side effects that accompany the administration ofa cell type in a particular patient. In determining the effective amountof cells to be administered in the treatment or prophylaxis of diseases,the physician should evaluate circulating plasma levels, and, in thecase of replacement therapy, the production of the gene product ofinterest.

Transduced cells are prepared for reinfusion according to establishedmethods. See, e.g., Abrahamsen, et al., J. Clin. Apheresis, vol.6:48-53(1991); Carter, et al., J. Clin. Apheresis, vol. 4:113-117(1988);Aebersold, et al., J. Immunol. Methods, vol. 112: 1-7(1988); Muul, etal., J. Immunol. Methods, vol. 101:171-181(1987), and Carter, et al.,Transfusion, vol. 27:362-365(1987). After a period of about 2-4 weeks inculture, the cells may number between 1×10⁶ and 1×10¹⁰. In this regard,the growth characteristics of cells vary from patient to patient andfrom cell type to cell type. About 72 hours prior to reinfusion of thetransduced cells, an aliquot is taken for analysis of phenotype andpercentage of cells expressing the therapeutic agent.

For administration, cells of the present invention can be administeredat a rate determined by the LD-50 of the cell type, and the side effectsof the cell type at various concentrations, as applied to the mass andoverall health of the patient. Administration can be accomplished viasingle or divided doses. Adult stem cells may also be mobilized usingexogenously administered factors that stimulate their production andegress from tissues or spaces, that may include, but are not restrictedto, bone marrow or adipose tissues. The exemplary glycoconjugates may beadministered concurrently, prior to and/or following stem cellsmobilization, or at a time when the amount of cells in the peripheralcirculation is optimal for the desired therapeutic endpoint.

EXAMPLES

Procedures

Intravenous cannulas were placed into the external jugular vein ofNOD-SCID mice under anesthesia (Institutional Animal Care and UseCommittee protocol #AM87046-07) to enable the efficient delivery of ¹¹¹In-labeled stem cells i.v. Tylenol elixir was administered by mouthafter recovery from anesthesia. Briefly, radiolabelled CD34+ cells weretaken up in 100-250 ul of 5% human plasma albumin in saline and injectedinto the cannula and then flushed with 50 ul of the albumin-saline. Themice were imaged by nuclear medicine.

Mice: NOD-SCID, female mice (non-obese diabetic/LtSz-scid/scid) wereobtained from the Jackson Laboratory, Bar Harbor, Me. at 1-2 months ofage. These animals were maintained in microisolator cages in a specialisolator room. The air was HEPA filtered, and the animals were changedin a laminar flow hood within the facility. All food, bedding, and waterwas sterilized. NOD-SCID mice were ideally suited for the study ofxenotransplanted tumors and hematopoietic cells and lymphocytes becauseof their immunoincompetence including greatly reduced NK activity. See,e.g. Hogan, et al., Biology of Blood & Marrow Transplantation, vol. 3:236-46 (1997); Noort, et al., Bone Marrow Transplantation, vol. 22 Suppl1: S58-60 (1998).

All administrations of agents or cells were done either i.v. or i.p.

Stem Cells: CD34+ stem cells were isolated from apheresis stem cellcollection products derived from deceased cancer patients. They werepurified to 95-99% purity using antibody conjugated to CD34 conjugatedto magnetics beads (MACS separation columns; Miltenyi Biotec, Auburn,Calif.) and cryopreserved.

Human mesenchymal stem cells (hMSCs; PT-2501) obtained through anFDA-monitored, paid bone marrow donor program were purchased fromPoietics Technologies, BioWhittaker (Walkerville, Md.). The cells werethawed according to manufacturer recommendations, resuspended, andradiolabelled in Mesenchymal Stem Cell Basal Medium (MSCBM).

Proteins administered: Orosomucoid (alpha-1 acid glycoprotein) andasialoorosomucoid (ASO) were administered in the following buffercontaining 0.16 mM Caprylate, 10 mM TRIS, 150 mM NaCl, pH 7.0.

Anesthesia and analgesia: A rodent anesthesia cocktail of 0.04 ml per20-30 g mouse i.p. (Rodent Cocktail recipe: 1.5 ml of 50 mg/ml ketamine,plus 1.5 ml of 20 mg/ml xylazine, plus 0.5 ml of 50 mg/ml acepromazine)was used. The anesthetic agent, Rodent anesthesia cocktail, wasadministered i.p. as follows:

1) for surgery: 0.04 ml per 20-30 g mouse; and

2) for imaging: 0.02 ml per 20-30 g mouse.

Post-surgical Analgesia: Tylenol 60 ul/20 g mouse (6.10 mg) wasadministered by mouth after anesthesia had partially worn off. Theanalgesic agent was tylenol by mouth at 60 ul (6.10 mg) per 20 g mouseimmediately after surgery or at the first signs of distress. Xylazinecontained in an anesthetic formulation may also act as an analgesic.

Surgical procedure (standard cannula placement): After anesthetizing theanimals as previously described, the threads for suturing a cannulafilled with citrate saline were soaked in 70% ethanol. The anesthetizedanimals were secured with paper tape on the operating platform ventralside up. The area from just below the clavicle to the ear was shaved.The shaven area was cleaned with Betadine and rinsed with 70% ethanol. Avertical incision was made in the skin of the right neck from the top ofthe rib cage to the jaw bone to expose the sternocleidomastoid musclewith the external jugular vein just beneath. To clearly expose theoperating field, the skin was retracted with wire hooks (secured tosmall weights). Retraction should not distort the underlying tissue butshould stabilize the area for visualization and cannula insertion. Thevein was cleared of overlying fat and fascia using microscopic forceps.The circulation in the superior vena cava was cut off using a half aknot of 4 O silk surgical sutures. One side of the thread was securedwith a clamped hemostat. A second piece of thread was looped around thebottom of the vein to make a half knot without pulling it tight. Thisloop was used to secure the cannula once it had been inserted into theexternal jugular vein. The surface of the vein was nicked with themicroscissors. The cannula was inserted into the vein with bevelled sideup. The cannula was slid down diagonally until the anchor was flush withthe wall of the vein and the lower knot tightened. The cannula wastested by pushing saline through it. The lower knot was finished afterverifying no leakage. A full knot was tied around the cannula using thetop thread. Saline flow in the cannula was monitored. The top thread wasused to go under, catching tissues, and a knot was tied over the cannulaagain with this thread. A full knot was made using an end of the topthread and the bottom thread. This secured the superior and inferiorthreads over the hub of the cannula to prevent accidental dislodgement.The cannula was clamped off and the syringe removed. The cannula waspositioned underneath the skin of the neck and exteriorized just belowthe occiput at the nape of the neck while rotating the animal (dorsalside up). An autoclip was used to staple the heat shrink part of thecannula in place near the exit. The cannula was cut to a reasonablelength (1.5-2.0 inches), and a wire plug was placed into it. The animalwas turned over to its original position and the neck closed with anautoclip, being careful not to puncture the cannula.

Surgical procedure (DaVinci Microport Vascular System cannulaplacement): The DaVinci Microport Vascular System (DaVinci Biomedical,South Lancaster, Mass.) is a closed injection route permitting itsimplantation up to 2 weeks prior to trafficking experiments without lossof patency. The essential difference is that the port is notexternalized as before. This eliminates additional risk forcontamination and damage to the cannula caused by chewing andscratching.

The incision area was cleaned with Betadine prior to initial cuts. Themouse was then taped (back side up) to the surgery board. An incision3-4 mm was made. Next, the incision was made on the chest 4-5 mm. Atunnel was made from the back incision to the front incision in order tofeed the cannula through the back to the chest. Heparin was pushedthrough the cannula. The cannula was then pulled through usinghemostats. The skin was pulled loose from the tissue on the back forplacement of the port. The port was sutured down to the tissue in themiddle upper neck area. It was sutured in two places using a triple knottie. Next, the mouse was turned on its back with its chest up. Thecannula was then cut at an angle, where at least 1 mm and at most 2 mmof cannula was inserted in the jugular vein. The jugular vein wasisolated in the chest after some fat and tissue was pulled away. Thearms of the mice were taped down on their sides because that pushes thechest forward and further exposes the jugular vein. Once the jugularvein was isolated, two sutures were placed around it. The top of thevein was tied off enough to slow the flow of blood, but not tocompletely stop the flow. The lower tie was one to 2 mm from the top,and it was not tightened. The lower tie was used later to hold thecannula in place and to stop excessive bleeding from the jugular vein.Next, a small cut was made in the jugular vein between the two ties, sothat the cannula could be fed into the vein. Once the cannula was placedin the vein the lower tie was tightened around the cannula within thevein. Next, the cannula was checked for leaks by running heparin throughthe cannula. After verifying no leaks, both incisions were closed.

¹¹¹Indium Oxine Labelling Procedure: ¹¹¹In-oxine labelling of adulthuman CD34+ or mesenchymal stem cells (hMSCs) was performed using amodification of the Amersham Healthcare Procedure for labellingautologous leukocytes.

Harvesting for tissues for histopathology: Tissues were harvested aftereuthanasia. After the 1-hour image, the organs were harvested and halfthe organ was fixed in 10% neutral buffered formalin and the other halfwas frozen in OTC for frozen sections. The images presented herein arefrom fixed tissues.

Necropsy Procedure For Collection of Mouse Tissues: An initial midlineskin incision from the anterior cervical region to the brim of the pubiswas made followed by an abdominal incision following linea alba from thesternum to the pubis with a lateral reflection of the abdominal wall byincision following the caudal ribs. The sternum was reflected anteriorlyby cutting the ribs at approximately the level of the costochrondraljunction, incising the diaphragm and pericardium as needed. Anteriorly,reflection of sternum was extended to include the ventral cervicalmuscles to expose the trachea. The trachea and esophagus were incised atthe mid cervical area and reflected caudally, cutting attachments asnecessary to remove the thoracic viscera in toto. Following removal ofthe thoracic viscera, the entire heart was dissected free and immersedin 10% neutral buffered formalin. After immersion, the heart wasmassaged lightly with serrated tissue forceps to force fixative into thecardiac chambers. The trachea with attached lung was then immersed infixative without further dissection. The spleen was visualized, omentalattachments incised, removed, and immersed whole in formalin fixative.The stomach and intestinal tract were removed by incising the rectum andreflecting the viscera anteriorly while cutting attachments asnecessary. The liver was removed in toto and immersed whole in formalinfixative. The kidneys were removed and immersed whole in formalinfixative. The pancreas was incised from the anterior duodenum andimmersed in formalin fixative.

Trimming of Tissues for Paraffin Processing and Microtomy: The heart wasplaced on the trimming board with the right ventricle on the uppersideand the left ventricle on the underside next to the trimming surface. Asingle upper-to-lower incision was made through the right ventricle andatrium and great vessels at the base of the heart continuing through theinterventricular septum and the left cardiac chambers to achieve twoapproximately equal halves. Each half was placed into separate embeddingcassettes containing fixative saturated foam pads and labelled “heart1”and “heart2”. The entire left and right lungs were separated frommidline tissues and placed flat on fixative-saturated foam pads incassettes labelled “left” and “right”lung. Liver sections were takenfrom the right lateral and medial liver lobes and placed into anappropriately labelled cassette. The left lateral and medial lobes weresectioned and handled in a similar manner. The entire spleen was placedin an appropriately labelled embedding cassette and oriented with onelong margin down, taking advantage of the curvature to increase initialsectional area. For one kidney, a whole coronal section was taken fromthe midpoint of the kidney. The remaining kidney was sectionedlongitudinally. Both sections were placed in a single cassette. Thecollected pancreas was placed on formalin-saturated foam pad in anappropriately labelled cassette.

Imaging procedures: Nuclear Medicine. NOD-SCID mice were anesthetizedusing rodent anesthesia cocktail. Once anesthetized, the mice wereplaced on a foam hemi-cylindrical mouse-positioning device (MPD,) andcovered with a tube sock. The MPD allows better visual separation of thelungs and liver as compared to placing the mouse on a flat surface. Thefoam on which the mouse was placed, and the tube sock coveringmaintained a comfortable temperature permitting longer imaging withoutadditional anesthesia. The MPD was placed on a narrow table between thedual heads of a Siemens E.Cam Gamma Camera and imaged statically ordynamically in 2-D or SPECT. ⁵⁷Co-Spot Marker is used to mark anatomicpositions (nose, tail, cannula, etc.). The data was analyzed using aSiemens ICON system for regions of interest or percent of injected dose(e.g., liver, spleen, heart).

CT Imaging: A CT scan was performed (G.E. Medical System High SpeedSpiral Tunnel) for tumor assessment and to enable theregistration/alignment of the nuclear medicine image with that of the CTin order to determine precise location of injected radiolabelled stemcells using the method described by Arata L., Clinical Uses for MedicalImage Registration: Experiences at Three Hospitals. Proceedings ofPACMEDTec Symposium in Honolulu, Hi., Aug. 17-21, 1998. and Nelson, etal., Electromedica 68 (2000) 45-48. CT scans were performed during anuclear medicine imaging session while the animals were underanesthesia. Anesthetized animals were transported to CT, either justprior to or immediately after, the nuclear medicine scan. Usually onlyone CT was done per animal. CT was used to precisely localize theradiolabelled materials anatomically, by fusing the CT image with thatof the nuclear medicine SPECT images.

Gamma camera imaging using a Siemens E.Cam dual head gamma cameramonitored the in vivo trafficking patterns of all human stem cellsdescribed in the following examples. Mice were placed on a MousePositioning Device (MPD) and placed between the detectors on the imagingplatform.

Example 1 ASO Administered Intravenously Directs Human CD34+ to theHeart

Asialoorosomucoid (ASO)/High Dose HSC: When an infusion of 5.75×10⁶ HSCwas preceded by 3.3 mg ASO, 77±1% of the infused cells were found in theheart immediately after infusion, 75±5% remained in the heart region at1.5 hr, decreasing to 52±1% at 24 hr.

5.75×10⁶, ¹¹¹In-labeled human CD34+ (hCD34+) peripheral blood stem cellswere administered intravenously (i.v.) via an external jugular veincannula to 2 month old, NOD-SCID, female mice (Non-obesediabetic/LtSz-scid/scid) obtained from the Jackson Laboratory, BarHarbor, Me. The radiolabelled CD34+ stem cells were administered afterpretreatment of the mouse with 3.3 mg of asialoorosomucoid (ASO) i.v.The in vivo trafficking patterns were followed by gamma camera imagingusing a Siemens E.Cam dual head gamma camera from immediately afterinjection up to 36 h post-infusion. Human CD34+ were isolated fromapheresis stem cell collection products derived from deceased cancerpatients. They were purified to 95-99% purity using antibody conjugatedto CD34 conjugated to magnetics beads (MACS separation columns, MiltenyiBiotec, Auburn, Calif.) and cryopreserved.

Radiolabelled CD34+ stem cells administered after ASO migratedimmediately to the heart. Anatomic localization was facilitated by theuse of a ⁵⁷Co-point source positioned at the level of the cannula. Up to79.2% of the injected dose was located in the heart at 1.5 hours. Thesecells did not migrate to the liver and spleen early in the post-infusionfollow up images but could be found in the liver later after 24 hours.However, 51.6-53.2% of the originally injected dose remained in theheart at 24 hours. At 36 hours imaging was conducted with the cannula invivo and with the cannula removed and placed next to the sacrificedanimal. These images show that the injected cells were not trapped inthe cannula but were actually in the heart.

Example 2 Orosomucoid Administered Intravenously Enables Human CD34+Cells to Migrate to the Liver and Spleen But Not to the Heart

Orosomucoid/High Dose HSC: When an infusion of 5.75×10⁶ HSC was precededby 5.5 mg orosomucoid, 74±3% of infused cells were found in the liverand spleen immediately after infusion, 74±4% of the cells remained inthe liver region at 1.5 hr, decreasing to 63±1% at 24 hr.

The preparation and procedures set forth in Example 1 were repeated.

5.75×10⁶, ¹¹¹In-labeled human CD34+ (hCD34+) peripheral blood stem cellswere administered intravenously (i.v.) via an external jugular veincannula to 2 month old, NOD-SCID, female mice (Non-obesediabetic/LtSz-scid/scid) obtained from the Jackson Laboratory, BarHarbor, Me. The radiolabelled CD34+ stem cells were administered afterpretreatment of the mouse with 5.5 mg of orosomucoid (0) i.v.

Mice were imaged and the biodistribution of the radiolabelled hCD34+cells monitored as described in Example 1. Radiolabelled hCD34+administered after 0 migrated immediately to the liver/spleen area andremained there until 36 hours. Anatomic localization was facilitated bythe use of a ⁵⁷Co-point source positioned at the level of the cannula.The localization to the liver/spleen region ranged from 76.3%immediately post-infusion to 63.6% at 24 hours. No ¹¹¹In-labeled cellswere found in the region of the heart.

At 36 hours imaging was conducted with the cannula in vivo and with thecannula removed and placed next to the sacrificed animal. These imagesshow that the injected cells were not trapped in the cannula.Radioactivity was found at or below the cannula placement, i.e., in theregion of the liver/spleen.

Example 3 Orosomucoid enables hCD34+ Cells to Migrate to theLiver/Spleen Without Significant Migration to the Heart

Orosomucoid/Low Dose HSC: When an infusion of 0.5×10⁶ HSC (one-tenth theprevious cell dose) was preceded by 11 mg orosomucoid, 43±2% of infusedcells were found in the liver and spleen immediately after infusion, and40±3% of the cells remained in the liver region at 1 hr.

The preparation and procedures set forth in Example 1 were repeated.0.5×10⁶ HSC, ¹¹¹In-labeled human CD34+ (hCD34+) peripheral blood stemcells were administered intravenously (i.v.) via an external jugularvein cannula to 2 month old, NOD-SCID, female mice (Non-obesediabetic/LtSz-scid/scid) obtained from the Jackson Laboratory, BarHarbor, Me. The radiolabelled CD34+ stem cells were administered afterpretreatment of the mouse with 11.0 mg of orosomucoid (0) i.v.

Mice were imaged and the biodistribution of the radiolabelled hCD34+cells monitored as described above. Approximately 1 hour after infusion,the mice were sacrificed and the organs were harvested, and half of theorgan was fixed in 10% neutral buffered formalin. Tissue sections wereexamined microscopically after immunohistochemical staining for humanCD34 and in situ hybridization for the visualization of human DNA.Nuclear medicine monitoring for the first ten minutes and 1 hourpost-infusion showed that the radiolabelled hCD34+ cells localized tothe region of the liver/spleen.

Microscopic examination of the heart after immunohistologic staining forCD34 demonstrated hCD34+ cells in the endocardial blood vessel. A fewhCD34+ cells could be seen in the lung in the alveolar septum. Clustersof cells with stem cell morphology could be seen in the hepaticsinusoid. In situ hybridization for human DNA clearly showed that hCD34+cells were not found in the heart muscle or interventricular septum butwere present in the lung.

Example 4 Asialoorosomucoid Followed by Orosomucoid Directs hCD34+ Cellsto the Heart and Lung But Not the Region of the Liver/Spleen

Asialoorosomucoid (ASO)+Orosomucoid (O)/Low Dose HSC: When infused ASOcaused HSC to localize in the heart, the protocol was changed to havethe ASO bolus chased with a bolus of orosomucoid to test whether theaccumulation in the heart would be maintained. HSC were againconcentrated in the heart when an infusion of 0.5×10⁶ HSC was precededby 3.3 mg ASO, then 5.5 mg orosomucoid. This caused 44±5% of the infusedcells to accumulate in the heart immediately after infusion. 37±3% ofthe infused cells remained in the heart region at 1 hr. The localizationin the heart was the major concentrated signal from the cells, althoughthe percent of infused was reduced from the ca. 75% seen in Example 1.

The preparation and procedures set forth in Example 1 were repeated.0.5×10⁶, In-labelled human CD34+ (hCD34+) peripheral blood stem cellswere administered intravenously (i.v.) via an external jugular veincannula to 2 month old, NOD-SCID, female mice (Non-obesediabetic/LtSz-scid/scid) obtained from the Jackson Laboratory, BarHarbor, Me. The radiolabelled CD34+ stem cells were administered afterpretreatment of the mouse with 3.3 mg of ASO i.v. followed by 5.5 mg 0i.v.

Mice were imaged and the biodistribution of the radiolabelled hCD34+cells monitored as described in Example 1. Nuclear medicine monitoringfor the first ten minutes and 1 hour post-infusion showed that theradiolabelled hCD34+ cells localized to the heart.

Approximately 1 hour after infusion, the mouse was sacrificed and theorgans were harvested and half the organ was fixed in 10% neutralbuffered formalin.

Microscopic examination of the heart after immunohistologic staining forCD34 revealed clusters of hCD34+ cells in the interventricular septum,and cells within those clusters that were morphologically similar to thestained cells but that were CD34 negative. These images reflected thebiodistribution depicted by nuclear medicine studies. The presence ofhCD34+ cells in the heart was dramatically demonstrated by in situhybridization. Both immunohistochemical staining for CD34 and in situhybridization for human DNA demonstrated that the infused stem cellslocalized to the lung and could be readily seen in the alveolar septa,blood vessels, and other structures. Detection of human DNA revealed thepresence of many more cells in the lung and heart than would have beenpredicted by CD34 staining. No hCD34+ cells or cells morphologicallyresembling hCD34+ cells were found in liver, spleen or kidney.

Example 5 HSC Administered in 5% Human Serum Albumin (WithoutOrosomucoid or Asialoorosomucoid) Migrated Predominantly to the Lungs

Plasma Albumin/High Dose HSC: When HSC were administered through thecatheter without prior protein infusion, 78±13% of infused cells werefound in the lungs at 0 hr. 54±10% at 1 hr, and 50±13% at 12 hr.Histological examination of lungs of mice similarly treated demonstratedinfused cells within the alveolar septa and the vasculature.

0.5×10⁶, ¹¹¹In-labeled HSC were administered intravenously (i.v.) via acannula implanted in the external jugular vein of a two-month old,female NOD-SCID mouse in 0.1 ml saline containing 5% human serumalbumin. Mice were imaged and the biodistribution of the radiolabelledhCD34+ cells monitored as described in Example 1.

Radiolabelled HSC, administered in saline containing 5% human serumalbumin, migrated immediately to the lungs. Anatomic localization of thelabelled cells was facilitated by the use of a ⁵⁷Co-point sourcepositioned at the level of the cannula exit site below the scapulae andnose. Moreover, the position marker at the cannula was verified to be atthe diaphragm by CT whole body scans, transverse and coronal sections.The clip at the cannula exit site served as a landmark. The lungs werevisualized below the nose marker and above the cannula marker and theliver and spleen below the cannula marker. Up to 95.4% of the injecteddose was located in the lungs at initial imaging (Table 1, below). Infour mice the values for the lungs ranged from 52.6-95.4% of whole bodyincorporation for the initial imaging time points. At 1 hour, HSC werelocated predominantly in the lungs with some counts visible in the bloodcirculation. In one mouse at 1 hour some localization was seen below thecannula marker, which may have been liver and spleen; however, theoutline was indistinct. At 12 hour in that mouse, radiolabelled CD34+stem cells were found in the liver/spleen region. However, more than34.7% (range 34.7-68.5%) of the originally injected dose remained in thelungs of other animals imaged at 12 hour.

While the localization to the lungs immediately after injection (initialor 0 hour time points) varied from animal to animal, the percent of theoriginal localization to the lungs remaining at subsequent scans wasmore constant. Using the dorsal images at 1 hour, 72.1-75.5% of thecells initially localized in the lung were retained in the lung region.Using the dorsal images at 12 hour, 78%, 72.1%, and 50.5% of the initiallung incorporation remained in the lungs of the three mice imaged.

Example 6 Orosomucoid Directs MSC to the Heart

Orosomucoid/Low Dose MSC: When a human mesenchymal stem cell infusion(0.56×10⁶ cells) was preceded by 11 mg orosomucoid, 68±7% of infusedcells were found in the heart at 0 hr, and 61±3% at 1 hr.

MSC were obtained from BioWhittaker, (Poietics Division, cryopreservedPT-2501 >750000 cells per ampoule) and labelled with ¹¹¹In as inprevious examples, except that the MSC were labelled, washed, andinjected in Basal Stem Cell Medium (Poietics) containing 5% human serumalbumin (HSA). 0.56×10⁶, ¹¹¹In-labelled, human mesenchymal stem cells(MSC) were administered via an implanted DaVinci Microport VascularSystem cannula in the external jugular vein of a two-month old, femaleNOD-SCID mouse in 0.21 ml of basal stem cell medium containing 5% humanserum albumin (HSA). Immediately prior to administration of MSC, 11.0 mgof orosomucoid was administered i.v. in 0.2 ml.

Mice were imaged and the biodistribution of the radiolabelled MSCs cellsmonitored as described in Example 1. Gamma camera monitoring initially(0 hr) and at 1 hr post-infusion showed that the radiolabelled MSClocalized to the region of the heart. Region of interest analysis of theimages revealed that approximately 61.7-75.5% of the injectedradioactivity initially localized to the heart and at 1 hr approximately58-64% of the infused cells remained in this region. The positions ofthe cannula, diaphragm, heart, lungs, and liver were verified by CTscans (coronal sections). In situ hybridization showed human cellspredominantly in the heart, but not the liver.

Example 7 Asialoorosomucoid Followed by Orosomucoid Directs MSC to theLiver/Spleen

MSC were obtained from BioWhittaker (Poietics Division, cryopreservedPT-2501>750000 cells per ampoule) and labelled with ¹¹¹In. As in Example6, the MSC were labelled, washed, and injected in Basal Stem Cell Medium(Poietics) containing 5% human serum albumin (HSA).

Asialoorosomucoid (ASO)+Orosomucoid/Low Dose MSC: This example wasdesigned to compare the trafficking of MSC with HSC (Example 4) at thelow cell dose, so the sequential infusion of ASO and orosomucoid used inExample 4 was applied. A human mesenchymal stem cell infusion (0.56×10⁶cells) was preceded by 4.3 mg ASO followed by 5.5 mg orosomucoid. 63±5%of the infused cells were found in the liver and spleen at 0 hr, and57±7% at 1 hr.

0.56×10⁶, ¹¹¹In-labeled MSC were administered i.v. in 0.21 ml of basalstem cell medium containing 5% human serum albumin (HSA). Prior toadministration of MSC, 0.1 ml containing 4.3 mg of ASO, followed by 0.1ml containing 5.5 mg orosomucoid were administered i.v. The ASO,orosomucoid and MSC were administered via an implanted DaVinci MicroportVascular system cannula in the external jugular vein of a two-month old,female NOD-SCID mouse.

Mice were imaged and the biodistribution of the radiolabelled MSCmonitored as in Example 1. Gamma camera monitoring initially and at 1 hrpost-infusion showed that the radiolabelled MSC localized to the regionof the liver/spleen. Region of interest analysis of the initial imagesrevealed that approximately 59.2-66.7% of the injected radioactivitylocalized to the liver/spleen and at 1 hr approximately 51.9-61.1% ofthe infused cells remained in this region.

The positions of the cannula, diaphragm, heart, lungs, and liver wereverified by CT scans. In situ hybridization confirmed the gamma camerabiodistribution data. Cells containing human DNA were foundpredominantly in the liver.

Results. The results of the experiments described above are summarizedin Table 1, below. TABLE 1 Stem Cells/Protein % Infused Cells in %Infused Cells Bolus Liver/Spleen In Heart % Infused Cells in LungsHSC/No Protein Bolus 78 ± 3% at 0 hr 54 ± 10% at 12 hr HSC/Orosomucoid74 ± 3% at 0 hr 74 ± 4% at 1.5 hr 63 ± 1% at 24 hr HSC/ASO 77 ± 1% at 0hr 75 ± 5% at 1.5 hr 52 ± 1% at 24 hr MSC/No Protein Bolus Saline only95% at 0 hr 87% at 1 hr 61% at 24 hr 59% at 48 hr  5% Human serum 94% at0 hr 87% at 1 hr 59% at 24 hr 57% at 48 hr RPMI-1640 95% at 0 hr 74% at1 hr 69% at 24 hr MSC/Orosomucoid 68 ± 7% at 0 hr 61 ± 3% at 12 hrMSC/ASO 63 ± 5% at 0 hr 57 ± 7% at 12 hr % Infused Cells in KidneyMSC/No Protein Bolus Saline only  4% at 1 hr 13% at 24 hr 14% at 48 hr 5% Human serum  2% at 1 hr 11% at 24 hr 14% at 48 hr RPMI-1640  7% at 1hr  9% at 24 hr

All publications, patents, patent applications, and other documentsmentioned in the specification are indicative of the level of thoseskilled in the art to which this invention pertains. All publications,patents, patent applications, and other documents are hereinincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application, orother document was specifically and individually indicated to beincorporated herein by reference in its entirety for all purposes.Subheadings in the specification document are included solely for easeof review of the document and are not intended to be a limitation on thecontents of the document in any way.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. In a method of administering stem cells to treat damaged heart orliver tissue in a subject, the improvement comprising administering tothe subject a composition comprising an asialoglycoprotein receptorblocker.
 2. The method of claim 1, wherein the asialoglycoproteinreceptor blocker is asialoorosomucoid.
 3. The method of claim 1, whereinthe asialoglycoprotein receptor blocker is selected from the groupconsisting of asialofetuin, a galactose-bonded polylysine, and agalactose-bonded polyglucoseamine.
 4. The method of claim 1, wherein theasialoglycoprotein receptor blocker is a glycoprotein comprising acarbohydrate structure selected from the group consisting ofcarbohydrate structures A, B, C, and D in FIG.
 3. 5. The method of claim4, wherein the glycoprotein is a human serum glycoprotein.
 6. The methodof claim 1, wherein the stem cell is selected from the group consistingof a mesenchymal stem cell, a hematopoietic stem cell, a CD34⁺mesenchymal stem cell, and a CD34⁺ hematopoietic stem cell.
 7. Themethod of claim 1, wherein the damaged tissue is heart.
 8. The method ofclaim 1, wherein the damaged tissue is liver.
 9. The method of claim 1,wherein the asialoglycoprotein receptor blocker is administered beforethe stem cell is administered.
 10. The method of claim 1, wherein theasialoglycoprotein receptor blocker is administered before and after thestem cell is administered.
 11. The method of claim 1, wherein theasialoglycoprotein receptor blocker is administered before the stem cellis administered during administration of the stem cell.
 12. The methodof claim 1, wherein the subject is a human subject.
 13. In a method ofadministering stems cells to treat damaged heart or liver tissue in asubject, the improvement comprising administering to the subject acomposition comprising orosomucoid.
 14. The method of claim 13, whereinthe stem cell is selected from the group consisting of a mesenchymalstem cell, a hematopoietic stem cell, a CD34⁺ mesenchymal stem cell, anda CD34⁺ hematopoietic stem cell.
 15. The method of claim 13, wherein thetarget tissue is heart.
 16. The method of claim 13, wherein the targettissue is liver.
 17. The method of claim 13, wherein the compositioncomprising orosomucoid is administered before the stem cells areadministered.
 18. The method of claim 13, wherein the compositioncomprising orosomucoid is administered before and after the stem cellsare administered.
 19. The method of claim 13, wherein the compositioncomprising orosomucoid is administered before the stem cells areadministered and during administration of the stem cells.
 20. The methodof claim 13, wherein the subject is a human subject.